The Egress of Fluid from the Brain via Arachnoid Transport: Foundational Work for the Tissue Engineering of the Arachnoid Granulation A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Cornelius Hoktsim Lam IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Allison Hubel Ph.D., advisor December 2011 i Cornelius Hoktsim Lam ©2011 ii Acknowledgements The instillation of the work ethic began early and I am most grateful to my father Dr. Chi-hung Lam and my late mother Yuen-chau Wong Lam for the upbringing that I had. In the last sixteen years, my wife Dr. Robin K. Solomon played a central role in my life and together with my boys help me complete what would otherwise be a very incomplete person. The production of this thesis is a culmination of decades of influences and experiences. While it may be argued that scientific inquiry begins very early in childhood, the formalization and education toward that goal require mentors who are understanding and compassionate toward the pupil continuously throughout his life. My mentors included Robert D. Coombe, PhD, current Chancellor of the University of Denver, Howard W. Blume, MD, PhD, Harvard University, and Abbas F. Sadikot, MD, PhD, McGill University. I have made mistakes, spent money, wasted time, and most importantly learned in their laboratories. Many factors must have entered into the decision for choosing a career in neurosurgery, and then years later for choosing neurotransport as a focus to study. Those who have guided me along this path are many. Department heads to fellow students and residents were all important to me. I would like to particularly thank the residents in the Department of Neurosurgery at the University of Minnesota from whom I derive much intellectual stimulation. I must thank my two chairmen under whom I have the privilege to work: Dr. Robert E. Maxwell, who understood what a slow learner is, and Dr. Stephen J. Haines, who although never explicitly encouraged me (he’s being practical), at every turn opened doors for me in the pursuit of this PhD. This dissertation would not be possible without Dr. Roderick A. Barke, Chief of Surgery at the Minneapolis VA Medical Center, who inspired and encouraged me, lobbied on my behalf, contributed to the laboratory equipment, and kept me on an even keel. I did not trim my practice in this pursuit and took great lengths to maintain the status quo, but I am certain that there was invariably some impact on my fellow colleagues in the Department of Neurosurgery at the University of Minnesota. I wish to thank all of them. i How this PhD began six years ago would be an interesting story, but not relevant in this section of the thesis. Suffice to say that Dr. Robert T. Tranquillo, Chairman of the Department of Biomedical Engineering, and Dr. Allison Hubel, my advisor, played important roles in this regard. They offered me an opportunity that not many people in my situation would have. At the same time, I wish to thank my PhD committee who guided me through these years. This work would not be possible without Eric A. Hansen, PhD, who essentially runs the Neurotransport laboratory. A Level ten Jedi in world of freshwater fishing, he educated me and my family in more ways than one. I would like to acknowledge the Augustine Foundation, the Institute of Engineering and Medicine, and the Regent Scholarship program for the financial and administrative support of this work. ii Dedication To my dearest boys, Zeke and Nate… I hope you never stop learning. iii Abstract The arachnoid tissue is a critical component for the removal of cerebrospinal fluid (CSF) and other substances. Failure results in hydrocephalus, increased intracranial pressure, and buildup of toxic materials in the brain. The purpose of this thesis is to establish a foundation for a biomimetic arachnoid construct. First, we characterized arachnoid cell transport in culture and on three-dimensional collagen scaffolds. Arachnoid cells were harvested from rat brainstems and cultured onto bilayered bovine collagen scaffolds. Cells exhibited arachnoid cell phenotype (positive for vimentin, desmoplakin, and cytokeratin), readily penetrated the collagen scaffold, and doubled approximately every 2–3 days. The transepithelial electrical resistance for a monolayer of cells was 160 Ω∙cm2, and permeability of indigo carmine was 6.7+1.1X10- 6 cm/s. Hydraulic conductivity of the collagen construct was 6.39 mL/min/mmHg/cm2. Because of practical limitations of primary culture which include slow growth, early senescence, and poor reproducibility, we created two immortalized rat arachnoid cell lines using retroviral gene transfer of SV40 large T antigen (SV40 LTAg) either with or without human telomerase (hTERT). They stably expressed either SV40 LTAg alone, or SV40 LTAg and hTERT, and demonstrated high proliferative rate, contact inhibition at confluence, and stable expression of protein markers characteristic of native arachnoid cells for more than 160 passages. We subsequently used them to determine arachnoidal barrier properties and paracellular transport. Permeabilities of urea, mannitol, and inulin were 2.9+1.1X10-6, 0.8+.18x10-6, and 1.0+.29x10-6 cm/s respectively. Size differential permeability testing with dextran clarified the arachnoidal blood-CSF-barrier limit and established a rate of intracellular transport to be two orders of magnitude slower than paracellular transport in a polyester membrane diffusion chamber. The theoretical pore size for paracellular space was 11Å and the occupancy to length ratios were 0.8 and 0.72 cm-1 for urea and mannitol respectively. The monolayer permeability was not significantly different from an apical to basal direction or vice versa. Gap junction may have a role in barrier formation. Although up-regulation of claudin by dexamethasone did not significantly alter paracellular transport, increasing intracellular cAMP decreased mannitol permeability. Calcium modulated paracellular transport, but only selectively with the ion chelator, EDTA, and with disruption of intracellular stores. Without the neurovascular unit of the blood-brain-barrier, the blood-CSF- barrier at the arachnoid tissue is anatomically and physiologically different from the vascular based blood-brain-barrier. These studies provide a three dimensional architecture, a stable iv cellular substrate, and baseline blood-CSF-barrier properties for the establishment of a viable bioartificial arachnoid shunt. v Table of Contents Acknowledgements........................................................................................................................... i Dedication ....................................................................................................................................... iii Abstract ........................................................................................................................................... iv Table of Contents ............................................................................................................................ vi List of Tables ................................................................................................................................... xi List of Figures ................................................................................................................................. xii List of Papers .................................................................................................................................. xiv Chapter 1: Background and Introduction ........................................................................................ 1 The arachnoid .............................................................................................................................. 2 History of arachnoid physiology research ............................................................................... 2 Embryology of arachnoid ......................................................................................................... 7 Anatomy of Arachnoid Tissue .................................................................................................. 8 Figure 1.1: Arachnoid granulation . ......................................................................................... 9 Table 1.1: Constituents in CSF of an adult (adapted from Greenberg[42]) ........................... 11 Figure 1.2: CSF flow ................................................................................................................ 12 Figure 1.3: The direction of movement of blood and debris from the intracranial cavity to the vascular system................................................................................................................ 13 Clinical Significance ................................................................................................................ 13 Figure 1.4: Blood washed into the CSF after trauma. ............................................................ 15 Barriers of the Brain and Neurotransport with Emphasis on the Arachnoid ............................ 16 Background ............................................................................................................................ 16 The arachnoid cell .................................................................................................................. 22 The arachnoid barrier ...........................................................................................................